ABSTRACT Dysregulation of intestinal iron absorption is a primary feature in iron overload disorders. Hypoxia-inducible factor 2? (HIF2?) is a critical regulator of iron absorption during increased systemic iron demands (i.e. iron deficiency, erythropoiesis, and pregnancy). HIF2? regulates expression of the apical iron transport machinery, divalent metal transporter 1 (DMT-1) and duodenal cytochrome b (Dcytb), and regulates the basolateral iron exporter ferroportin independent of hepcidin, the master systemic regulator of iron homeostasis. Disrupting intestinal HIF2? decreases tissue iron accumulation in iron overload disorders, such as ?-thalassemia and heredity hemochromatosis. Moreover, in ?-thalassemia, disruption of intestinal HIF2? also improves anemia. This has laid the foundation for HIF2?-based therapeutics for ?-thalassemia and heredity hemochromatosis, an area actively being researched by Peloton Therapeutics. Although our results demonstrate a central role for HIF2? in intestinal iron absorption, the underlying mechanisms behind its overlapping and distinct roles in iron deficiency, ?-thalassemia, and heredity hemochromatosis are still unclear. We hypothesize that a decrease in systemic hepcidin triggers HIF2? activation in intestinal epithelia, leading to an iron-absorptive response, which is critical for tissue iron accumulation in iron overload disorders. Our long-term goals are to improve existing HIF2?-based therapies and identify novel HIF2?-based therapies in iron-related disorders. The major goal of this proposal is to assess the precise mechanisms by which HIF2? selectively regulates iron absorption and determine if inhibition of HIF2? signaling and downstream mediators can be used to restrict tissue iron overload. We will pursue our objectives through three interconnected Specific Aims. Aim 1 will identify mechanisms leading to rapid activation of HIF2? in hereditary hemochromatosis. Our data suggest a crosstalk between the systemic iron regulator, hepcidin, and intestinal HIF2?. This concept will be tested in novel mouse models that allow us to acutely and temporally regulate the hepcidin-ferroportin axis. Aim 2 will characterize precise mechanisms leading to an iron-absorptive HIF2? response. We have identified mothers against decapentaplegic homolog 3 (SMAD3) as a novel factor that is essential for expression of iron- absorptive (but not glycolytic, angiogenic, or inflammatory) HIF2? target genes. We will elucidate the specific role and underlying mechanisms behind SMAD3 regulation of HIF2? signaling. Aim 3 will assess the requirement for HIF2?-induced intestinal ferritinophagy in systemic iron homeostasis and iron overload disorders. Nuclear coactivator-4 (NCOA4), the major regulator of ferritinophagy, is directly regulated by HIF2? and is highly induced in iron deficiency, ?-thalassemia, and heredity hemochromatosis. We will examine the role that autophagic ferritin turnover plays in iron absorption using novel mouse models. Together, the proposed in vivo and in vitro studies will identify fundamental roles of HIF2? in iron absorption and iron overload disorders and lay the foundation for pursuing new therapeutic strategies targeting HIF2?.